This invention relates generally to nucleic acid hybridization techniques, and more particularly to methods for preparing and applying DNA probes.
Nucleic acid probes have widespread applications in molecular biology, medicine, and biotechnology, e.g., Johnson, "DNA Reassociation and RNA Hybridization of Bacterial Nucleic Acids," Methods in Microbiology, Vol. 18, pgs. 33-74 (1985); Petersson et al., "Nucleic Acid Hybridization--An Alternative Tool in Diagnostic Microbiology,"Immunology Today, Vol. 6, pgs 268-272 (1985). The probes comprise labeled fragments of single stranded or double stranded DNA or RNA which contain base sequences that are complementary to sequences of interest on target DNAs (or RNAs). Probes are applied to target nucleic acids under conditions which allow the probe to anneal to tne complementary sequence on the target. whenever douole stranded probes or target nucleic acids are involved, they must be converted to single stranded form by heating, or other means, to permit the prooe to anneal to the target. The probe's location on the target, or the location of the target itself, is detected by a label carried by the probe.
Probes are prepared either by cloning and labeling nucleic acids having sequences complementary to those on the target sought to be identified, e.g., via nick-translation, Rigby et al., J. Mol. Biol., Vol. 113, pgs 237-251 (1977), or the like, or by synthesizing labeled oligonucleotides of the appropriate sequence, e.g. Chollet et al., Nucleic Acids Research, Vol. 13, pgs. 1529-1541 (1985), or wallace et al., Nucleic Acids Research, Vol. 9, pgs 879-894 (1981). In either case probes are prepared independently of any steps for preparing the target nucleic acid for application of the probe. Advantages and disadvantages are associated with each method of constructing probes depending on the particular application contemplated.
One technique for which probe preparation and manner of application is especially important is in situ hybridization. Here target nucleic acids remain in their natural biological settings, e.g., DNA or RNA in chromosomes or nuclei, or in mRNA in cytoplasm. Typically the probes are directed to target sequences which are present in very low copy numbers so that signal-to-noise problems are critical, Angerer et al., "In situ Hybridization to Cellular RNAs," in Setlow and Hollaender, Eds., Genetic Engineering, Vol. 7, pgs. 43-65 (1985). As applied to in situ hybridization, current methods of probe construction and application have several drawbacks. whenever double stranded probes are used (or even when single stranded probes are used in some cases) conditions which promote annealing of probe to target also promote annealing of the complementary strands of the probe. The effective concentration of the probe is reduced. If probe concentration is increased to compensate for this effect, background noise increases because of nonspecific and/or mismatched binding of the excess probe.
Current techniques of preparing the target chromosomes or nuclei aim to denature all of the target DNA. whether single stranded or double stranded probe is used, significant mismatcn binding of probe to target can occur because virtually the entire target nucleic acid is denatured and available for hybridization, not just the regions which contain complementary sequences to the probe. Finally, currently used conditions for promoting denaturation are often excessively disruptive to the substrate or biological structure containing the target nucleic acids.
In view of the foregoing it would be desirable to have an alternative procedure for preparing and applying nucleic acid probes which overcame some of the drawbacks associated with current hybridization techniques.